ARTICLE Retinoids,
also known as vitamin A analogs, have been shown to stimulate wound healing
[1]. Faster reepithelialization with topical application of tretinoin was
reported in corneal abrasions [2, 3] as well as enhancement of the repairing
process in the photodamaged skin of hairless mice [4]. In addition, use
of tretinoin before dermabrasion [5] and superficial electroepilation-induced
wounds [6] was found to increase the healing phenomenon.
Adapalene is a new synthetic drug with strong retinoid agonist pharmacology.
This stable and less irritating formulation was developed for the topical
treatment of acne vulgaris [7]. While adapalene is known to behave similarly
to tretinoin pharmacologically, we propose it might have a role in enhancing
wound repair. Thus, this study was planned to evaluate the efficacy of
adapalene in full-thickness wound healing, to compare and discuss the
effects of tretinoin as well as collagenase, which also contribute to
the wound repairing process.
Material and methods
Thirty-two adult male Wistar-Albino rats, weighing between 170 and 340
g which were obtained from the animal laboratory of Suleyman Demirel University
Faculty of Medicine, were used in the study. Guidelines for the humane
use of laboratory animals were strictly adhered to throughout the study.
Experimental setting
Animals were housed in a 20° C room and fed rat chow and water
ad libitum. The rats were anesthetized with intraperitoneally injected
ketamine 10 mg/kg and xylazine 4 mg/kg. After shaving, two circular, full-thickness
wounds were made for each animal under sterile conditions with a standard
8-mm dermatologic punch biopsy, on both sides of the midline on the back.
The animals were randomly allocated into four groups. No treatment was
given to Group I rats (n:8) which served as the control group. Tretinoin
cream (0.1%) was applied topically once a day in Group II (n:8), adapalene
gel (0.1%) in Group III (n:8), and collagenase ointment in Group IV (n:8)
in the same manner (Table
I). The drugs were applied directly to both of the wounds of each
animal. Treatment was started the day after the wound was created (day
1) and continued up to 7 days for both wounds of each rat. The wounds
were cleaned with saline-moistened swabs to remove any crust. Erythema
and erosion of skin surrounding the wound was noted as a reaction of irritation.
On day 7, the wounds were photographed from 30 cm by Canon EOS500 camera
to measure the wound surface area using AutoCat v3.0 (Autodesk Inc.) program.
The wounds on the left side of each animal were excised on day 7, for
histopathologic and biochemical analysis. Excision was performed leaving
the wound in the center covering the entire wound base. Topical treatments
were continued for the right side-wounds up to 14 days and the same procedure
was repeated on day 14.
Biochemical analysis
Hydroxyproline (HP) levels were measured in dry tissue modifying the
method described by Woessner [8].
Biopsy specimens
The specimen was prepared for sectioning by routine histopathologic
methods. For each tissue sample on the 7th and 14th days, 6
to 25 sections were taken for histopathologic evaluation. Hematoxylin-eosin
(HE), periodic acid schiff (PAS), Giemsa, Masson's trichrome and Verhoeff's
elastic stains were applied. Silver impregnation for reticulum was also
performed. Epithelialization, neutrophil, lymphocyte, histiocyte, fibroblast
and eosinophil infiltration, angiogenesis and vascular endothelial proliferation
as well as mast cell counting in 5 high power field under the light microscope
were examined. Collagen fibers were carefully classified as to whether
they were closely packed as thick bundles or a loose network of thin fibers.
The evaluation was described as 0 for none, 1 for mild, 2 for moderate
and 3 for severe type findings. The pathologist was blinded to the study
protocol.
Statistical analysis
Treatment groups were compared with controls in all aspects. Differences
among groups were evaluated by using Kruskal-Wallis Analysis of Variance
Test and pairwise comparisons were done by Mann Whitney-U Test. Values
were expressed as median (min-max) and a p value < 0.05 was accepted
as statistically significant.
Results
Regarding measurement of wound surface area which was calculated by
using AutoCat program, there was no statistically significant reduction
among any of the groups at day 7 or 14. All of the wounds were incompletely
healed by day 14 with no scar formation (Fig.
1). However, we observed that the surrounding skin of most of the
tretinoin (0.1%) cream-treated wounds was irritated.
HP levels of the groups are shown in Table
II. At day 7, a significant decrease was detected in Group II compared
to the control group (p = 0.018). HP results revealed no difference neither
in Group III nor in Group IV versus control.
At day 14, there was a statistically significant enhancement in HP levels
of Group II (p = 0.002), although no difference was detected in Groups
III and IV when compared to the control group.
Histopathologic examination revealed that findings of improved healing
were more remarkable in Groups II and III than in the other groups. In
Group II, epithelialization and granulation tissue formation were evident
and collagen fibers formed dense and thick bundles towards the end of
the healing process although reticular and elastic fibers were less prominent.
Angiogenesis and mast cell counts were viewed similarly in Group III.
Cellular infiltration and angiogenesis were obvious at day 7 in Group
III (Fig. 2a) compared
to the control group. Neutrophils and lymphocytes were predominant in
the cellulary infiltrate at day 7 and were decreased at the end of day
14. Vascular and mast cell proliferation continued up to the 14th day.
Reticular and elastic fibers were easily detected throughout the healing
period (Fig. 2b). Collagen
fibers were closely packed (Fig.
2c).
There was no clear histopathologic difference between Group IV and the
control group in terms of cellular reactions and angiogenesis. However,
collagen fibers were prominent in Group IV.
Discussion
In this controlled animal study, the effects of three currently used
topical preparations on full-thickness wound healing were compared. There
has been conflicting information about retinoic acid derivatives in the
literature, whether they improved or impaired wound healing [1, 4, 9,
10. Moreover, the new naphthoic acid derivative adapalene, possessing
similar pharmacological properties to retinoic acid [7], has not previously
been investigated in the wound repairing process.
Full-thickness punch model has been widely used and wound contraction
was assessed as an additional parameter for healing [11]. Using the full-thickness
punch model, our results revealed no difference between any of the treated-wound
and control groups in relation to wound diameters and surface area. This
might be partially due to the fact that vitamin A does not affect the
initial contraction phase [12]. Moreover, it is obvious that wound contraction
need not show concordance with qualitative healing.
Vitamin A analogs are known to alter epithelial differentiation and
proliferation in a dose-dependent manner [3, 4]. Reported research on
retinoids revealed discrepancies between the studies [4, 13-15]. In an
in vitro study by Daly et al. [13] reduction in collagen
synthesis and cellular proliferation with vitamin A derivatives was confirmed.
Otley et al. [14] suggested that neither preoperative nor postoperative
topical application of tretinoin benefited wound healing. The detrimental
effect of tretinoin on epithelial healing when applied to fresh partial-thickness
wounds has also been described previously [16].
On the other hand, topical application of tretinoin cream has been reported
to increase fibroblast activation, collagen and elastin formation and
neovascularization [4, 15]. Pretreatment with tretinoin cream has been
reported to enhance reepithelialization of partial [16] and full-thickness
wounds in animals [12]. In addition, retinoids significantly reverse the
inhibitory effects of steroids on wound healing and stimulate granulation
tissue [17-19]. Lack of correlation between in vitro and in
vivo studies was partly due to the highly complicated environment
of wounds in animals [11].
Levenson et al. [20] investigated the healing of rat skin wounds
and found abundant collagen formation during the second week of wounding
with continued enhancement up to 6 or 7 weeks. Histopathological findings
in the present study showed the positive effect of tretinoin cream on
epithelialization, granulation tissue formation and angiogenesis. Collagen
bundles were more evident towards the end of post-wounded period. A significant
decrease of HP levels at day 7 in Group II compared to the control group
turned into the highest levels at day 14. Despite the fact that type I
collagen was not evaluated in this study, this delayed overproduction
of collagen might suggest that tretinoin cream (0.1%) has a more significant
effect on the synthesis of type I collagen [21-24] which plays an important
role on the later stages of wound healing [25]. This suggestion might
be supported with the dominance of thick collagen bundles indicating type
I collagen versus indefinite reticular fibers which mostly contain
collagen type III [26, 27]. It has also been reported that the effect
of vitamin A was most pronounced in the final epithelial phase of the
healing period [12]. In the study of Prutkin, vitamin A derivatives directly
applied to open circular wounds, were found to stimulate wound healing
[1]. This finding was confirmed by Smith et al. in an open incision-al
wound model [18]. In contrast, using the same model, Golan et al.
[9] could not detect an improvement in wound healing with the topical
use of vitamin A. Recently, in another animal study, postoperative application
of tretinoin to open lesions was reported to result in a significant retardation
of reepithelialization [10]. In the present study, it was noted that tretinoin
cream application caused irritation of the surrounding skin which reduced
the quality of the healing process [14].
Adapalene has been reported to produce similar or greater pharmacological
activity compared to retinoic acids. Adapalene modulates differentiation
in cells and inhibits proliferation [7, 28]. In spite of its antiinflammatory
function, adapalene has been reported to have positive effects on the
survival of reconstructed skin system [28]. In addition, it was shown
that adapalene 0.1% gel was not irritating due to its unique receptor
specificity [29]. Our results revealed that adapalene increased cellular
infiltration, granulation tissue and angiogenesis. Moreover, collagen,
reticular and elastic fibers, which strengthen tension and structural
maintenance, were prominently enhanced. The 7th and 14th day HP levels
did not reveal any difference between adapalene and the control group
at day 7, whereas enhanced levels were pronounced at day 14. This might
be suggested as a positive but late onset effect of adapalene on wound
improvement, as in the tretinoin cream-treated group. Moreover, we did
not observe any sign of irritation with topical adapalene. The associated
preservative in both adapalene gel and tretinoin cream was methyl parahydroxybenzoate
(Table I) which might
be responsible for strong cellular infiltration resulting in inflammatory
changes and epithelial alterations [30]. This suggestion needs to be confirmed
by further studies by application of different preservative agents in
these formulations. In addition, different forms of cream and gel preparations
of the formulations used in this study could change penetration capacity.
Thus, aqueous gel of adapalene might have stayed less time than tretinoin
cream on the wounded area. It may also be proposed that adapalene gel
is an agreable formulation with the potential of resorption by rats through
open wounds.
In the present study, increased HP levels by collagenase ointment was
demonstrated although the difference did not reach a statistically significant
level. Histopathologically, collagen fibers were detected more prominently
than in the control group. The HP results in adapalene and collagenase-treated
wounds did not reveal significance, although different from the controls.
Nevertheless, evident histopathological findings of improved healing made
us consider whether this difference could be clarified by studies comprising
larger sample sizes.
CONCLUSION
In conclusion, tretinoin and adapalene contributed to wound healing by
enhancing collagen production, neovascularization and granulation tissue
formation. Retinoid dermatitis described previously [14] due to topical
tretinoin formulations has been a handicap for usage of these drugs as
was also observed in the present experiment. Adapalene seems to have a
later effect, but its continued effect on collagen was prominent and it
is better tolerated than tretinoin. Nevertheless, further studies determining
collagen subtypes in different wound models are necessary to clarify its
effects on wound healing.
This study was not sponsored by any of the medical firms.
Article accepted on 25/10/01
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